Despite recent progress with engineered T cell therapies in the treatment of cancer, resistance to the activity of these cells remains a significant hurdle to their success. Increased potency, greater penetration of solid tumors, and mechanisms to defeat the immunosuppressive tumor microenvironment are still needed for many cancer types. To address this problem, we propose an innovative strategy that overcomes tumor resistance by modifying CAR T cells to constitutively express an enzyme that synthesizes a potent cytotoxic chemotherapy drug from a non-toxic prodrug at the cancer cell surface or into the tumor microenvironment. The selective and local elaboration of potent anti-neoplastic drugs at the tumor site may overcome the immune resistance to the cells, and tumor escape by antigen loss variants, as the drug does not rely on tumor antigen expression and may diffuse locally. These live and regulatable anticancer agents will serve as targetable micropharmacies that synthesize the drug itself, not simply deliver a payload. We term them ?Synthetic Enzyme-Activated KillER? cells or SEAKER cells. SEAKER cells will combine the innate tumor killing ability of CAR T cells with the specific activation of a cytotoxic drug at the tumor site. The aims of this project are to design and construct various SEAKER cell systems and validate their functions in vitro, and to then test the efficacy and pharmacological properties of the SEAKER systems in various animal tumor models. CAR T constructs targeting the well-established CD19 tumor antigen or the ubiquitous Wilm's tumor protein will be engineered to express the carboxypeptidase CPG2, which can activate a cytotoxic agent modified with an inhibitory glutamate residue that renders it non-toxic. In the presence of CPG2, the inhibitory glutamate on the prodrug will be cleaved and the drug will become active. Various enzyme/prodrug systems will be developed and tested in vitro, and optimal conditions for prodrug activation and constitutive CPG2 expression will be determined. A cell-surface anchored form and a secreted form of CPG2 will be engineered. The optimized SEAKER constructs will be used to generate CAR-T cells from primary human T cells, and infused into various xenogeneic murine tumor models. Pharmacokinetics and in-vivo expansion of the SEAKER cells will be measured. The ability of the SEAKER cells to shrink tumor mass and extend survival of tumor-engrafted mice will be assessed and compared to traditional CAR-T cells. Distribution of prodrug versus activated drug will be examined using LCMS/MS technology. These studies will indicate the feasibility and efficacy of this novel and promising approach to advancing cellular therapies in cancer treatment.